The Toll-like receptors (TLRs) comprise a family of at least 10 integral membrane receptors that are essential for initiating inflammatory reactions in response to pathogens such as viruses and bacteria. The major objective of this research is to elucidate the structures of external domains of TLRs in order to learn how they recognize pathogen associated molecular patterns (PAMP). We have developed a baculovirus expression system for the TLRs, in conjunction with the Protein Expression Lab [SAIC] and with Joseph Shiloach, head of the NIDDK Biotechnology Unit. The purification scheme for the target TLRs utilizes a double affinity tag and typically yields 10-15 mg of highly purified sample/10L prep. [unreadable] The molecular structure of the ectodomain of TLR3 which recognizes dsRNA has been determined. The structure is horseshoe shaped with 23 leucine-rich repeats but differs from most leucine-rich repeat proteins in having no twist. The extensive beta sheet concave surface forms a platform for two insertions and 11 N-linked glycans and suggests several possible RNA binding sites. We are continuing attempts to locate the dsRNA binding site by crystallography.[unreadable] We have conducted a mutational analysis to provide information about the probable binding site for ds RNA.Several mutations in adjacent site completely abrogate binding and provide clear indication of the binding site.